Unactivated oocytes as cytoplast recipients for nuclear transfer

ABSTRACT

A method of reconstituting an animal embryo involves transferring a diploid nucleus into an oocyte which is arrested in the metaphase of the second meiotic division. The oocyte is not activated at the time of transfer, so that the donor nucleus is kept exposed to the recipient cytoplasm for a period of time. The diploid nucleus can be donated by a cell in either the G0 or G1 phase of the cell cycle at the time of transfer. Subsequently, the reconstituted embryo is activated. Correct ploidy is maintained during activation, for example, by incubating the reconstituted embryo in the presence of a microtubule inhibitor such as nocodazole. The reconstituted embryo may then give rise to one or more live animal births. The invention is useful in the production of transgenic animals as well as non-transgenics of high genetic merit.

[0001] This invention relates to the generation of animals including butnot being limited to genetically selected and/or modified animals, andto cells useful in their generation.

[0002] The reconstruction of mammalian embryos by the transfer of adonor nucleus to an enucleated oocyte or one cell zygote allows theproduction of genetically identical individuals. This has clearadvantages for both research (i.e. as biological controls) and also incommercial applications (i.e. multiplication of genetically valuablelivestock, uniformity of meat products, animal management).

[0003] Embryo reconstruction by nuclear transfer was first proposed(Spemann, Embryonic Development and Induction 210-211 Hofner PublishingCo., New York (1938)) in order to answer the question of nuclearequivalence or ‘do nuclei change during development?’. By transferringnuclei from increasingly advanced embryonic stages these experimentswere designed to determine at which point nuclei became restricted intheir developmental potential. Due to technical limitations and theunfortunate death of Spemann these studies were not completed until1952, when it was demonstrated in the frog that certain nuclei coulddirect development to a sexually mature adult (Briggs and King, Proc.Natl. Acad. Sci. USA 38 455-461 (1952)). Their findings led to thecurrent concept that equivalent totipotent nuclei from a singleindividual could, when transferred to an enucleated egg, give rise to“genetically identical” individuals. In the true sense of the meaningthese individuals would not be clones as unknown cytoplasmiccontributions in each may vary and also the absence of any chromosomalrearrangements would have to be demonstrated.

[0004] Since the demonstration of embryo cloning in amphibians, similartechniques have been applied to mammalian species. These Techniques fallinto two categories: 1) transfer of a donor nucleus to a maturedmetaphase II oocyte which has had its chromosomal DNA removed and 2)transfer of a donor nucleus to a fertilised one cell zygote which hashad both pronuclei removed. In ungulates the former procedure has becomethe method of choice as no development has been reported using thelatter other than when pronuclei are exchanged.

[0005] Transfer of the donor nucleus into the oocyte cytoplasm isgenerally achieved by inducing cell fusion. In ungulates fusion isinduced by application of a DC electrical pulse across thecontact/fusion plane of the couplet. The same pulse which induces cellfusion also activates the recipient oocyte. Following embryoreconstruction further development is dependent on a large number offactors including the ability of the nucleus to direct development i.e.totipotency, developmental competence of the recipient cytoplast (i.e.oocyte maturation), oocyte activation, embryo culture (reviewed Campbelland Wilmut in Vth World Congress on Genetics as Applied to Livestock 20180-187 (1994)).

[0006] In addition to the above we have shown that maintenance ofcorrect ploidy during the first cell cycle of the reconstructed embryois of major importance (Campbell et al., Biol. Reprod. 49 933-942(1993); Campbell et al., Biol. Reprod. 50 1385-1393 (1994)). During asingle cell cycle all genomic DNA must be replicated once and only onceprior to mitosis. If any of the DNA either fails to replicate or isreplicated more than once then the ploidy of that nucleus at the time ofmitosis will be incorrect. The mechanisms by which replication isrestricted to a single round during each cell cycle are unclear,however, several lines of evidence have implicated that maintenance ofan intact nuclear membrane is crucial to this control. The morphologicalevents which occur in the donor nucleus after transfer into anenucleated metaphase II oocyte have been studied in a number of speciesincluding mouse (Czolowiska et al., J. Cell Sci. 69 19-34 (1984)),rabbit (Collas and Robl, Biol. Reprod. 45 455-465 (1991)), pig (Pratheret al., J. Exp. Zool. 225 355-358 (1990)), cow (Kanka et al., Mol.Reprod. Dev. 29 110-116 (1991)). Immediately upon fusion the donornuclear envelope breaks down (NEBD), and the chromosomes prematurelycondense (PCC). These effects are catalysed by a cytoplasmic activitytermed maturation/mitosis/meiosis promoting factor (MPF). This activityis found in all mitotic and meiotic cells reaching a maximal activity atmetaphase. Matured mammalian oocytes are arrested at metaphase of the2nd meiotic division (metaphase II) and have high MPF activity. Uponfertilisation or activation MPF activity declines, the second meioticdivision is completed and the second polar body extruded, the chromatinthen decondenses and pronuclear formation occurs. In nuclear transferembryos reconstructed when MPF levels are high NEBD and PCC occur; theseevents are followed, when MPF activity declines, by chromatindecondensation and nuclear reformation and subsequent DNA replication.In reconstructed embryos correct ploidy can be maintained in one of twoways; firstly by transferring nuclei at a defined cell cycle stage, e.g.diploid nuclei of cells in G1, into metaphase II oocytes at the time ofactivation; or secondly by activating the recipient oocyte andtransferring the donor nucleus after the disappearance of MPF activity.In sheep this latter approach has yielded an increase in the frequencyof development to the blastocyst stage from 21% to 55% of reconstructedembryos when using blastomeres from 16 cell embryos as nuclear donors(Campbell et al., Biol. Reprod. 50 1385-1393 (1994)).

[0007] These improvements in the frequency of development ofreconstructed embryos have as yet not addressed the question of nuclearreprogramming. During development certain genes become “imprinted” i.e.are altered such that they are no longer transcribed. Studies onimprinting have shown that this “imprinting” is removed during germ cellformation (i.e. reprogramming). One possibility is that thisreprogramming is affected by exposure of the chromatin to cytoplasmicfactors which are present in cells undergoing meiosis. This raises thequestion of how we may mimic this situation during the reconstruction ofembryos by nuclear transfer in order to reprogram the developmentalclock of the donor nucleus.

[0008] It has now been found that nuclear transfer into an oocytearrested in metaphase II can give rise to a viable embryo if normalploidy (i.e. diploidy) is maintained and if the embryo is not activatedat the time of nuclear transfer. The delay in activation allows thenucleus to remain exposed to the recipient cytoplasm.

[0009] According to a first aspect of the present invention there isprovided a method of reconstituting an animal embryo, the methodcomprising transferring a diploid nucleus into an oocyte which isarrested in the metaphase of the second meiotic division withoutconcomitantly activating the oocyte, keeping the nucleus exposed to thecytoplasm of the recipient for a period of time sufficient for thereconstituted embryo to become capable of giving rise to a live birthand subsequently activating the reconstituted embryo while maintainingcorrect ploidy. At this stage, the reconstituted embryo is a singlecell.

[0010] In principle, the invention is applicable to all animals,including birds such as domestic fowl, amphibian species and fishspecies. In practice, however, it will be to non-human animals,especially non-human mammals, particularly placental mammals, that thegreatest commercially useful applicability is presently envisaged. It iswith ungulates, particularly economically important ungulates such ascattle, sheep, goats, water buffalo, camels and pigs that the inventionis likely to be most useful, both as a means for cloning animals and asa means for generating transgenic animals. It should also be noted thatthe invention is also likely to be applicable to other economicallyimportant animal species such as, for example, horses, llamas orrodents, e.g. rats or mice, or rabbits.

[0011] The invention is equally applicable in the production oftransgenic, as well as non-transgenic animals. Transgenic animals may beproduced from genetically altered donor cells. The overall procedure hasa number of advantages over conventional procedures for the productionof transgenic (i.e. genetically modified) animals which may besummarised as follows:

[0012] (1) fewer recipients will be required;

[0013] (2) multiple syngeneic founders may be generated using clonaldonor cells;

[0014] (3) subtle genetic alteration by gene targeting is permitted;

[0015] (4) all animals produced from embryos prepared by the inventionshould transmit the relevant genetic modification through the germ lineas each animal is derived from a single nucleus; in contrast, productionof transgenic animals by pronuclear injection or chimerism afterinclusion of modified stem cell populations by blastocyst injectionproduces a proportion of mosaic animals in which all cells do notcontain the modification and may not transmit the modification throughthe germ line; and

[0016] (5) cells can be selected for the site of genetic modification(e.g. integration) prior to the generation of the whole animal.

[0017] It should be noted that the term “transgenic”, in relation toanimals, should not be taken to be limited to referring to animalscontaining in their germ line one or more genes from another species,although many transgenic animals will contain such a gene or genes.Rather, the term refers more broadly to any animal whose germ line hasbeen the subject of technical intervention by recombinant DNAtechnology. So, for example, an animal in whose germ line an endogenousgene has been deleted, duplicated, activated or modified is a transgenicanimal for the purposes of this invention as much as an animal to whosegerm line an exogenous DNA sequence has been added.

[0018] In embodiments of the invention in which the animal istransgenic, the donor nucleus is genetically modified. The donor nucleusmay contain one or more transgenes and the genetic modification may takeplace prior to nuclear transfer and embryo reconstitution. Althoughmicro-injection, analogous to injection into the male or femalepronucleus of a zygote, may be used as a method of genetic modification,the invention is not limited to that methodology: mass transformation ortransfection techniques can also be used e.g. electroporation, viraltransfection or lipofection.

[0019] In the method of the invention described above, a diploid nucleusis transferred from a donor into the enucleated recipient oocyte. Donorswhich are diploid at the time of transfer are necessary in order tomaintain the correct ploidy of the reconstituted embryo; thereforedonors may be either in the G1 phase or preferably, as is the subject ofour co-pending PCT patent application No. PCT/GB96/02099 filed today(claiming priority from GB 9517780.4), in the G0 phase of the cellcycle.

[0020] The mitotic cell cycle has four distinct phases, G, S, G2 and M.The beginning event in the cell cycle, called start, takes place in theG1 phase and has a unique function. The decision or commitment toundergo another cell cycle is made at start. Once a cell has passedthrough start, it passes through the remainder of the G1 phase, which isthe pre-DNA synthesis phase. The second stage, the S phase, is when DNAsynthesis takes place. This is followed by the G2 phase, which is theperiod between DNA synthesis and mitosis. Mitosis itself occurs at the Mphase. Quiescent cells (which include cells in which quiescence has beeninduced as well as those cells which are naturally quiescent, such ascertain fully differentiated cells) are generally regarded as not beingin any of these four phases of the cycle; they are usually described asbeing in a G0 state, so as to indicate that they would not normallyprogress through the cycle. The nuclei of quiescent G0 cells, like thenuclei of G1 cells, have a diploid DNA content; both of such diploidnuclei can be used in the present invention.

[0021] Subject to the above, it is believed that there is no significantlimitation on the cells that can be used in nuclear donors: fully orpartially differentiated cells or undifferentiated cells can be used ascan cells which are cultured in vitro or abstracted ex vivo. The onlylimitation is that the donor cells have normal DNA content and bekaryotypically normal. A preferred source of cells is disclosed in ourco-pending PCT patent application No. PCT/GB95/02095, published as WO96/07732. It is believed that all such normal cells contain all of thegenetic information required for the production of an adult animal. Thepresent invention allows this information to be provided to thedeveloping embryo by altering chromatin structure such that the geneticmaterial can re-direct development.

[0022] Recipient cells useful in the invention are enucleated oocyteswhich are arrested in the metaphase of the second meiotic division. Inmost vertebrates, oocyte maturation proceeds in vivo to this fairly latestage of the egg maturation process and then arrests. At ovulation, thearrested oocyte is released from the ovary (and, if fertilisationoccurs, the oocyte is naturally stimulated to complete meiosis). In thepractice of the invention, oocytes can be matured either in vitro or invivo and are collected on appearance of the 1st polar body or as soon aspossible after ovulation, respectively.

[0023] It is preferred that the recipient be enucleate. While it hasbeen generally assumed that enucleation of recipient oocytes in nucleartransfer procedures is essential, there is no published experimentalconfirmation of this judgement. The original procedure described forungulates involved splitting the cell into two halves, one of which waslikely to be enucleated (Willadsen Nature 320 (6) 63-65 (1986)). Thisprocedure has the disadvantage that the other unknown half will stillhave the metaphase apparatus and that the reduction in volume of thecytoplasm is believed to accelerate the pattern of differentiation ofthe new embryo (Eviskov et al., Development 109 322-328 (1990)).

[0024] More recently, different procedures have been used in attempts toremove the chromosomes with a minimum of cytoplasm. Aspiration of thefirst polar body and neighbouring cytoplasm was found to remove themetaphase II apparatus in 67% of sheep oocytes (Smith & Wilmut Biol.Reprod. 40 1027-1035 (1989)). Only with the use of DNA-specificfluorochrome (Hoechst 33342) was a method provided by which enucleationwould be guaranteed with the minimum reduction in cytoplasmic volume(Tsunoda et al., J. Reprod. Fertil. 82 173 (1988)). In livestockspecies, this is probably the method of routine use at present (Prather& First J. Reprod. Fertil. Suppl. 41 125 (1990), Westhusin et al., Biol.Reprod. (Suppl.) 42 176 (1990)).

[0025] There have been very few reports of non-invasive approaches toenucleation in mammals, whereas in amphibians, irradiation withultraviolet light is used as a routine procedure (Gurdon Q. J. Microsc.Soc. 101 299-311 (1960)). There are no detailed reports of the use ofthis approach in mammals, although during the use of DNA-specificfluorochrome it was noted that exposure of mouse oocytes to ultravioletlight for more than 30 seconds reduced the developmental potential ofthe cell (Tsunoda et al., J. Reprod. Fertil. 82 173 (1988)).

[0026] As described above enucleation may be achieved physically, byactual removal of the nucleus, pro-nuclei or metaphase plate (dependingon the recipient cell), or functionally, such as by the application ofultraviolet radiation or another enucleating influence.

[0027] After enucleation, the donor nucleus is introduced either byfusion to donor cells under conditions which do not induce oocyteactivation or by injection under non-activating conditions. In order tomaintain the correct ploidy of the reconstructed embryo the donornucleus must be diploid (i.e. in the G0 or G1 phase of the cell cycle)at the time of fusion.

[0028] Once suitable donor and recipient cells have been prepared, it isnecessary for the nucleus of the former to be transferred to the latter.Most conveniently, nuclear transfer is effected by fusion. Activationshould not take place at the time of fusion.

[0029] Three established methods which have been used to induce fusionare:

[0030] (1) exposure of cells to fusion-promoting chemicals, such aspolyethylene glycol;

[0031] (2) the use of inactivated virus, such as Sendai virus; and

[0032] (3) the use of electrical stimulation.

[0033] Exposure of cells to fusion-promoting chemicals such aspolyethylene glycol or other glycols is a routine procedure for thefusion of somatic cells, but it has not been widely used with embryos.As polyethylene glycol is toxic it is necessary to expose the cells fora minimum period and the need to be able to remove the chemical quicklymay necessitate the removal of the zona pellucida (Kanka et al., Mol.Reprod. Dev. 29 110-116 (1991)). In experiments with mouse embryos,inactivated Sendai virus provides an efficient means for the fusion ofcells from cleavage-stage embryos (Graham Wistar Inst. Symp. Monogr. 919 (1969)), with the additional experimental advantage that activationis not induced. In ungulates, fusion is commonly achieved by the sameelectrical stimulation that is used to induce parthogenetic activation(Willadsen Nature 320 (6) 63-65 (1986), Prather et al., Biol. Reprod. 37859-866 (1987)). In these species, Sendai virus induces fusion in aproportion of cases, but is not sufficiently reliable for routineapplication (Willadsen Nature 320 (6) 63-65 (1986)).

[0034] While cell-cell fusion is a preferred method of effecting nucleartransfer, it is not the only method that can be used. Other suitabletechniques include microinjection (Ritchie and Campbell, J. Reproductionand Fertility Abstract Series No. 15, p60).

[0035] In a preferred embodiment of the invention, fusion of the oocytekaryoplast couplet is accomplished in the absence of activation byelectropulsing in 0.3M mannitol solution or 0.27M sucrose solution;alternatively the nucleus may be introduced by injection in a calciumfree medium. The age of the oocytes at the time of fusion/injection andthe absence of calcium ions from the fusion/injection medium preventactivation of the recipient oocyte.

[0036] In practice, it is best to enucleate and conduct the transfer ssoon as possible after the oocyte reaches metaphase II. The time thatthis will be post onset of maturation (in vitro) or hormone treatment(in vivo) will depend on the species. For cattle or sheep, nucleartransfer should preferably take place within 24 hours; for pigs, within48 hours; mice, within 12 hours; and rabbits within 20-24 hours.although transfer can take place later, it becomes progressively moredifficult to achieve as the oocyte ages. High MPF activity is desirable.

[0037] Subsequently, the fused reconstructed embryo, which is generallyreturned to the maturation medium, is maintained without being activatedso that the donor nucleus is exposed to the recipient cytoplasm for aperiod of time sufficient to allow the reconstructed embryo to becomecapable, eventually, of giving rise to a live birth (preferably of afertile offspring).

[0038] The optimum period of time before activation varies from speciesto species and can readily be determined by experimentation. For cattle,a period of from 6 to 20 hours is appropriate. The time period shouldprobably not be less than that which will allow chromosome formation,and it should not be so long either that the couplet activatesspontaneously or, in extreme cases that it dies.

[0039] When it is time for activation, any conventional or othersuitable activation protocol can be used. Recent experiments have shownthat the requirements for parthogenetic activation are more complicatedthan had been imagined. It had been assumed that activation is anall-or-none phenomenon and that the large number of treatments able toinduce formation of a pronucleus were all causing “activation”. However,exposure of rabbit oocytes to repeated electrical pulses revealed thatonly selection of an appropriate series of pulses and control of theCa²⁺ was able to promote development of diploidized oocytes tomid-gestation (Ozil Development 109 117-127 (1990)). Duringfertilization there are repeated, transient increases in intracellularcalcium concentration (Cutbertson & Cobbold Nature 316 541-542 (1985))and electrical pulses are believed to cause analogous increases incalcium concentration. There is evidence that the pattern of calciumtransients varies with species and it can be anticipated that theoptimal pattern of electrical pulses will vary in a similar manner. Theinterval between pulses in the rabbit is approximately 4 minutes (OzilDevelopment 109 117-127 (1990)), and in the mouse 10 to 20 minutes(Cutbertson & Cobbold Nature 316 541-542 (1985)), while there arepreliminary observations in the cow that the interval is approximately20 to 30 minutes (Robl et al., in Symposium on Cloning Mammals byNuclear Transplantation (Seidel ed.), Colorado State University, 24-27(1992)). In most published experiments activation was induced with asingle electrical pulse, but new observations suggest that theproportion of reconstituted embryos that develop is increased byexposure to several pulses (Collas & Robl Biol. Reprod. 43 877-884(1990)). In any individual case, routine adjustments may be made tooptimise the number of pulses, the field strength and duration of thepulses and the calcium concentration of the medium.

[0040] In the practice of the invention, correct ploidy must bemaintained during activation. It is desirable to inhibit or stabilisemicrotubule polymerisation in order to prevent the production ofmultiple pronuclei, thereby to maintain correct ploidy. This can beachieved by the application of a microtubule inhibitor such asnocodazole at an effective concentration (such as about 5 μg/ml).Colchecine and colcemid are other microtubule inhibitors. Alternatively,a microtubule stabiliser, such as, for example, taxol could be used.

[0041] The molecular component of microtubules (tubulin) is in a stateof dynamic equilibrium between the polymerised and non-polymerisedstates. Microtubule inhibitors such as nocodazole prevent the additionof tubulin molecules to microtubules, thereby disturbing the equilibriumand leading to microtubule depolymerisation and destruction of thespindle. It is preferred to add the microtubule inhibitor a sufficienttime before activation to ensure complete, or almost complete,depolymerisation of the microtubules. Twenty to thirty minutes is likelyto be sufficient in most cases. A microtubule stabiliser such as taxolprevents the breakdown of the spindle and may also therefore prevent theproduction of multiple pronuclei. Use of a microtubule stabiliser ispreferably under similar conditions to those used for microtubuleinhibitors.

[0042] The microtubule inhibitor or stabiliser should remain presentafter activation until pronuclei formation. It should be removedthereafter, and in any event before the first division takes place.

[0043] In a preferred embodiment of the invention at 30-42 hours postonset of maturation (bovine and ovine, i.e. 6-18 hours post nucleartransfer) the reconstructed oocytes are placed into medium containingnocodazole (5 μg/ml) and activated using conventional protocols.Incubation in nocodazole may be continued for 4-6 hours following theactivation stimulus (dependent upon species and oocyte age).

[0044] According to a second aspect of the invention, there is provideda viable reconstituted animal embryo prepared by a method as describedpreviously.

[0045] According to a third aspect of the invention, there is provided amethod of preparing an animal, the method comprising:

[0046] (a) reconstituting an animal embryo as described above; and

[0047] (b) causing an animal to develop to term from the embryo; and

[0048] (c) optionally, breeding from the animal so formed.

[0049] Step (a) has been described in depth above.

[0050] The second step, step (b) in the method of this aspect of theinvention is to cause an animal to develop to term from the embryo. Thismay be done directly or indirectly. In direct development, thereconstituted embryo from step (a) is simply allowed to develop withoutfurther intervention beyond any that may be necessary to allow thedevelopment to take place. In indirect development, however, the embryomay be further manipulated before full development takes place. Forexample, the embryo may be split and the cells clonally expanded, forthe purpose of improving yield.

[0051] Alternatively or additionally, it may be possible for increasedyields of viable embryos to be achieved by means of the presentinvention by clonal expansion of donors and/or if use is made of theprocess of serial (nuclear) transfer. A limitation in the presentlyachieved rate of blastocyst formation may be due to the fact that amajority of the embryos do not “reprogram” (although an acceptablenumber do). If this is the case, then the rate may be enhanced asfollows. Each embryo that does develop itself can be used as a nucleardonor at the 32-64 cell stage; alternatively, inner cell mass cells canbe used at the blastocyst stage. If these embryos do indeed reflectthose which have reprogrammed gene expression and those nuclei are infact reprogrammed (as seems likely), then each developing embryo may bemultiplied in this way by the efficiency of the nuclear transferprocess. The degree of enhancement likely to be achieved depends uponthe cell type. In sheep, it is readily possible to obtain 55% blastocyststage embryos by transfer of a single blastomere from a 16 cell embryoto a preactivated “Universal Recipient” oocyte. So it is reasonable tohypothesise that each embryo developed from a single cell could giverise to eight at the 16 cell stage. Although these figures are just arough guide, it is clear that at later developmental stages the extentof benefit would depend on the efficiency of the process at that stage.

[0052] Aside from the issue of yield-improving expediencies, thereconstituted embryo may be cultured, in vivo or in vitro to blastocyst.

[0053] Experience suggests that embryos derived by nuclear transfer aredifferent from normal embryos and sometimes benefit from or even requireculture conditions in vivo other than those in which embryos are usuallycultured (at least in vivo). The reason for this is not known. Inroutine multiplication of bovine embryos, reconstituted embryos (many ofthem at once) have been cultured in sheep oviducts for 5 to 6 days (asdescribed by Willadsen, In Mammalian Egg Transfer (Adams, E. E., ed.)185 CRC Press, Boca Raton, Fla. (1982)). In the practice of the presentinvention, though, in order to protect the embryo it should preferablybe embedded in a protective medium such as agar before transfer and thendissected from the agar after recovery from the temporary recipient. Thefunction of the protective agar or other medium is twofold: first, itacts as a structural aid for the embryo by holding the zona pellucidatogether; and secondly it acts as barrier to cells of the recipientanimal's immune system. Although this approach increases the proportionof embryos that form blastocysts, there is the disadvantage that anumber of embryos may be lost.

[0054] If in vitro conditions are used, those routinely employed in theart are quite acceptable.

[0055] At the blastocyst stage, the embryo may be screened forsuitability for development to term. Typically, this will be done wherethe embryo is transgenic and screening and selection for stableintegrants has been carried out. Screening for non-transgenic geneticmarkers may also be carried out at this stage. However, because themethod of the invention allows for screening of donors at an earlierstage, that will generally be preferred.

[0056] After screening, if screening has taken place, the blastocystembryo is allowed to develop to term. This will generally be in vivo. Ifdevelopment up to blastocyst has taken place in vitro, then transferinto the final recipient animal takes place at this stage. If blastocystdevelopment has taken place in vivo, although in principle theblastocyst can be allowed to develop to term in the pre-blastocyst host,in practice the blastocyst will usually be removed from the (temporary)pre-blastocyst recipient and, after dissection from the protectivemedium, will be transferred to the (permanent) post-blastocystrecipient.

[0057] In optional step (c) of this aspect of the invention, animals maybe bred from the animal prepared by the preceding steps. In this way, ananimal may be used to establish a herd or flock of animals having thedesired genetic characteristic(s).

[0058] Animals produced by transfer of nuclei from a source ofgenetically identical cells share the same nucleus, but are not strictlyidentical as they are derived from different oocytes. The significanceof this different origin is not clear, but may affect commercial traits.Recent analyses of the mitochondrial DNA of dairy cattle in the IowaState University Breeding Herd revealed associated with milk andreproductive performance (Freeman & Beitz, In Symposium on CloningMammals by Nuclear Transplantation (Seidel, G. E. Jr., ed.) 17-20,Colorado State University, Colorado (1992)). It remains to be confirmedthat similar effects are present throughout the cattle population and toconsider whether it is possible or necessary in specific situations toconsider the selection of oocytes. In the area of cattle breeding theability to produce large numbers of embryos from donors of high geneticmerit may have considerable potential value in disseminating geneticimprovement through the national herd. The scale of application willdepend upon the cost of each embryo and the proportion of transferredembryos able to develop to term.

[0059] By way of illustration and summary, the following scheme sets outa typical process by which transgenic and non-transgenic animals may beprepared. The process can be regarded as involving five steps:

[0060] (1) isolation of diploid donor cells;

[0061] (2) optionally, transgenesis, for example by transfection withsuitable constructs, with or without selectable markers;

[0062] (2a) optionally screen and select for stable integrants—skip formicro-injection;

[0063] (3) embryo reconstitution by nuclear transfer;

[0064] (4) culture, in vivo or in vitro, to blastocyst;

[0065] (4a) optionally screen and select for stable integrants—omit ifdone at 2a—or other desired characteristics;

[0066] (5) transfer if necessary to final recipient.

[0067] This protocol has a number of advantages over previouslypublished methods of nuclear transfer:

[0068] 1) The chromatin of the donor nucleus can be exposed to themeiotic cytoplasm of the recipient oocyte in the absence of activationfor appropriate periods of time. This may increase the “reprogramming”of the donor nucleus by altering the chromatin structure.

[0069] 2) Correct ploidy of the reconstructed embryo is maintained whenG0/G1 nuclei are transferred.

[0070] 3) Previous studies have shown that activation responsiveness ofbovine/ovine oocytes increases with age. One problem which haspreviously been observed is that in unenucleated aged oocytesduplication of the meiotic spindle pole bodies occurs and multipolarspindles are observed. However, we report that in embryos reconstructedand maintained with high MPF levels although nuclear envelope breakdownand chromatin condensation occur no organised spindle is observed. Theprematurely condensed chromosomes remain in a tight bunch, therefore wecan take advantage of the ageing process and increase the activationresponse of the reconstructed oocyte without adversely affecting theploidy of the reconstructed embryo.

[0071] According to a fourth aspect of the invention, there is providedan animal prepared as described above.

[0072] Preferred features of each aspect of the invention are as foreach other aspect, mutatis mutandis.

[0073] The invention will now be described by reference to theaccompanying Examples which are provided for the purposes ofillustration and are not to be construed as being limiting on thepresent invention. In the following description, reference is made tothe accompanying drawing, in which:

[0074]FIG. 1 shows the rate of maturation of bovine oocytes in vitro.

EXAMPLE 1 “MAGIC” Procedure Using Bovine Oocytes

[0075] Recipient oocytes the subject of this experimental procedure aredesignated MAGIC (Metaphase Arrested G1/G0 AcceptIng Cytoplast)Recipients.

[0076] The nuclear and cytoplasmic events during in vitro oocytematuration were studied. In addition the roles of fusion and activationin embryos reconstructed at different ages were also investigated. Thestudies have shown that oocyte maturation is asynchronous; however, apopulation of matured oocytes can be morphologically selected at 18hours (FIG. 1).

Morphological Selection of Oocytes

[0077] In FIG. 1 ovaries were obtained from a local abattoir andmaintained at 28-32° C. during transport to the laboratory. Cumulusoocyte complexes (COC's) were aspirated from follicles 3-10 mm indiameter using a hypodermic needle (1.2 mm internal diameter) and placedinto sterile plastic universal containers. The universal containers wereplaced into a warmed chamber (35° C.) and the follicular materialallowed to settle for 10-15 minutes before pouring off three quarters ofthe supernatant. The remaining follicular material was diluted with anequal volume of dissection medium (TCM 199 with Earles salts (Gibco),75.0 mg/l kanamycin, 30.0 mM Hepes, pH 7.4, osmolarity 280 mOsmols/KgH₂O) supplemented with 10% bovine serum, transferred into an 85 mm petridish and searched for COC's under a dissecting microscope.

[0078] Complexes with at least 2-3 compact layers of cumulus cells wereselected washed three times in dissection medium and transferred intomaturation medium (TC medium 199 with Earles salts (Gibco), 75 mg/lkanamycin, 30.0 mM Hepes, 7.69 mM NaHCO₃, pH 7.8, osmolarity 280mOsmols/Kg H₂O) supplemented with 10% bovine serum and 1×10⁶ granulosacells/ml and cultured on a rocking table at 39° C. in an atmosphere of5% CO₂ in air. Oocytes were removed from the maturation dish and wetmounted on ethanol cleaned glass slides under coverslips which wereattached using a mixture of 5% petroleum jelly 95% wax. Mounted embryoswere then fixed for 24 hours in freshly prepared methanol: glacialacetic acid (3:1), stained with 45% aceto-orcein (Sigma) and examined byphase contrast and DIC microscopy using a Nikon Microphot-SA, the graphin FIG. 1 shows the percentage of oocytes at MII and those with avisible polar body.

Activation of Bovine Follicular Oocytes

[0079] If maturation is then continued until 24 hours these oocytesactivate at a very low rate (24%) in mannitol containing calcium (Table1a). However, removal of calcium and magnesium from the electropulsingmedium prevents any activation.

[0080] Table 1a shows activation of bovine follicular oocytes matured invitro for different periods. Oocytes were removed from the maturationmedium, washed once in activation medium, placed into the activationchamber and given a single electrical pulse of 1.25 kV/cm for 80 μs.TABLE 1a No. Hours post onset of Pronuclear formation of oocytes (N)maturation (hpm) (age hrs)) (% activation) 73 24 24.6 99 30 84.8 55 4592.7*

Activation Response of Sham Enucleated Bovine Oocytes

[0081] Table 1b shows activation response of in vitro matured bovineoocytes sham enucleated at approximately 22 hours post onset ofmaturation (hpm). Oocytes were treated exactly as for enucleation, asmall volume of cytoplasm was aspirated not containing the metaphaseplate. After manipulation the oocytes were given a single DC pulse of1.25 Kv/cm and returned to the maturation medium, at 30 hpm and 42 hpmgroups of oocytes were mounted, fixed and stained with aceto-orcein. Theresults show the number of oocytes at each time point from fiveindividual experiments as the number of cells having pronuclei withrespect to the total number of cells. TABLE 1b No. cells having No.cells having pronuclei/Total pronuclei/Total no. of cells no. of cellsEXPERIMENT 30 hpm 42 hpm 1 1/8 — 2 0/24 0/30 3 0/21 0/22 4 0/27 0/25 50/19 0/1

Pronuclear Formation in Enucleated Oocytes

[0082] Table 2 shows pronuclear formation in enucleated oocytes fused toprimary bovine fibroblasts (24 hpm) and subsequently activated (42 hpm).The results represent five separate experiments. Oocytes were dividedinto two groups, group A were incubated in nocodazole for 1 hour priorto activation and for 6 hours following activation. Group B were nottreated with nocodazole. Activated oocytes were fixed and stained withaceto-orcein 12 hours post activation. The number of pronuclei (PN) ineach parthenote was then scored under phase contrast. The results areexpressed as the percentage of activated oocytes containing 1 or morepronuclei. TABLE 2 TOTAL 1 PN 2 PN 3 PN 4 PN >4 PN GROUP A 52 100 0 0 00 GROUP B 33 45.2 25.8 16.1 3.2 9.7

[0083] The absence of an organised spindle and the absence of a polarbody suggests that in order to maintain ploidy in the reconstructedembryo then only a diploid i.e. G0/G1 nucleus should be transferred intothis cytoplasmic situation. Incubation of activated oocytes in thepresence of the microtubule inhibitor nocodazole for 5 hours, 1 hourprior to and following the activation stimulus prevents the formation ofmicronuclei (Table 2) and thus when the donor nucleus is in the G0/G1phase of the cell cycle the correct ploidy of the reconstructed embryois maintained.

Results

[0084] These results show that:

[0085] i) these oocytes can be enucleated at 18 hours post onset ofmaturation (FIG. 1);

[0086] ii) enucleated oocytes can be fused to donor blastomeres/cells ineither 0.3M mannitol or 0.27M sucrose alternatively the donor the cellsor nuclei can be injected in calcium free medium in the absence of anyactivation response;

[0087] iii) reconstructed embryos or enucleated pulsed oocytes can becultured in maturation medium and do not undergo spontaneous activation;

[0088] iv) the transferred nucleus is seen to undergo nuclear envelopebreakdown (NEBD) and chromosome condensation. No organisedmeiotic/mitotic spindle is observed regardless of the cell cycle stageof the transferred nucleus;

[0089] v) such manipulated couplets will activate at 30 hours and 42hours with a frequency equal to unmanipulated control oocytes;

[0090] vi) no polar body is observed following subsequent activation,regardless of the cell cycle stage of the transferred nucleus;

[0091] viii) upon subsequent activation 1-5 micronuclei are formed perreconstructed zygote (Table 2).

Reconstruction of Bovine Embryos Using “MAGIC” Procedure

[0092] In preliminary experiments this technique has been applied to thereconstruction of bovine embryos using primary fibroblasts synchronisedin the G0 phase of the cell cycle by serum starvation for five days. Theresults are summarised in Table 3.

[0093] Table 3 shows development of bovine embryos reconstructed bynuclear transfer of serum starved (G0) bovine primary fibroblasts intoenucleated unactivated MII oocytes. Embryos were reconstructed at 24 hpmand the fused couplets activated at 42 hpm. Fused couplets wereincubated in nocodazole (5 μg/ml) in M2 medium for 1 hour prior toactivation and 5 hours post activation. Couplets were activated with asingle DC pulse of 1.25 KV/cm for 80 μsec. TABLE 3 NUMBER OFBLASTOCYSTS/ TOTAL NUMBER OF FUSED EXPERIMENT NUMBER COUPLETS %BLASTOCYSTS 1 1/30 3.3 2 4/31 12.9

EXAMPLE 2 “MAGIC” Procedure Using Ovine Oocytes

[0094] Similar observations to those in Example 1 have also been made inovine oocytes which have been matured in vivo. Freshly ovulated oocytescan be retrieved by flushing from the oviducts of superstimulated ewes24 hours after prostaglandin treatment. The use of calcium magnesiumfree PBS/1.0% FCS as a flushing medium prevents oocyte activation.Oocytes can be enucleated in calcium free medium and donor cellsintroduced as above in the absence of activation. No organised spindleis observed, multiple nuclei are formed upon subsequent activation andthis may be suppressed by nocodazole treatment.

Results

[0095] In preliminary experiments in sheep, a single pregnancy hasresulted in the birth of a single live lamb. The results are summarisedin Tables 4 and 5.

[0096] Table 4 shows development of ovine embryos reconstructed bytransfer of an embryo derived established cell line to unactivatedenucleated in vivo matured ovine oocytes. Oocytes were obtained fromsuperstimulated Scottish blackface ewes, the cell line was establishedfrom the embryonic disc of a day 9 embryo obtained from a Welsh mountainewe. Reconstructed embryos were cultured in the ligated oviduct of atemporary recipient ewe for 6 days, recovered and assessed fordevelopment. TABLE 4 NUMBER OF MORULA, BLA DATE OF STOCYSTS/ NUCLEARPASSAGE TOTAL TRANSFER NUMBER NUMBER 17.1.95 6  4/28 19.1.95 7  1/1031.1.95 13  0/2  2.2.95 13  0/14  7.2.95 11  1/9  9.2.95 11  1/2 14.2.9512 16.2.95 13  3/13 TOTAL 10/78 (12.8%)

[0097] Table 5 shows induction of pregnancy following transfer of allmorula/blastocyst stage reconstructed embryos to the uterine horn ofsynchronised final recipient blackface ewes. The table shows the totalnumber of embryos from each group transferred the frequency of pregnancyin terms of ewes and embryos, in the majority of cases 2 embryos weretransferred to each ewe. A single twin pregnancy was established whichresulted in the birth of a single live lamb. TABLE 5 PASSAGE NUMBER“MAGIC” P6 4 P7 1 P11 2 P12 0 P13 3 TOTAL MOR/BL 10 TOTAL NUMBER EWES 6PREGNANT EWES 1 (16.7) % FOETUSES/ 2/10 (20.0) TOTAL TRANSFERRED (%)

1. A method of reconstituting an animal embryo, the process comprisingtransferring a diploid nucleus into an oocyte which is arrested in themetaphase of the second meiotic division without concomitantlyactivating the oocyte, keeping the nucleus exposed to the cytoplasm ofthe recipient for a period of time sufficient for the embryo to becomecapable of giving rise to a live birth and subsequently activating thereconstituted embryo while maintaining correct ploidy.
 2. A method asclaimed in claim 1, in which the animal is an ungulate species.
 3. Amethod as claimed in claim 2, in which the animal is a cow or bull, pig,goat, sheep, camel or water buffalo.
 4. A method as claimed in any oneof claims 1 to 3, in which the donor nucleus is genetically modified. 5.A method as claimed in any one of claims 1 to 4, wherein the diploidnucleus is donated by a quiescent cell.
 6. A method as claimed in anyone of claims 1 to 5, wherein the recipient oocyte is enucleate.
 7. Amethod as claimed in any one of claims 1 to 6, wherein nuclear transferis achieved by cell fusion.
 8. A method as claimed in any one of claims1 to 7, wherein the animal is a cow or bull and wherein the donornucleus is kept exposed to the recipient cytoplasm for a period of from6 to 20 hours prior to activation.
 9. A method as claimed in any one ofclaims 1 to 8, wherein correct ploidy is maintained during activation bymicrotubule inhibition.
 10. A method as claimed in claim 9, whereinmicrotubule inhibition is achieved by the application of nocodazole. 11.A method as claimed in any one of claims 1 to 8, wherein correct ploidyis maintained during activation by microtubule stabilisation.
 12. Amethod as claimed in claim 11, wherein microtubule stabilisation isachieved by the application of taxol.
 13. A method of preparing ananimal, the method comprising: (a) reconstituting an animal embryo asclaimed in any preceding claim; (b) causing an animal to develop to termfrom the embryo; and (c) optionally, breeding from the animal so formed.14. A method as claimed in claim 13, wherein the animal embryo isfurther manipulated prior to full development of the embryo.
 15. Amethod as claimed in claim 14, wherein more than one animal is derivedfrom the embryo.
 16. A reconstituted animal embryo which is capable ofgiving rise to a live birth and is prepared by a method as claimed inany one of claims 1 to
 12. 17. An animal prepared by a method as claimedin any one of claims 13 to
 15. 18. An animal developed from an embryo asclaimed in claim 16.